Sugarcane (Saccharum spp.), a major cash crop that is an important source of sugar and bioethanol, is strongly influenced by the impacts of biotic and abiotic stresses. The intricate polyploid and aneuploid genome of sugarcane has shown various limits for conventional breeding strategies. Nonetheless, biotechnological engineering currently offers the best chance of introducing commercially significant agronomic features. In this study, an efficient Agrobacterium-mediated transformation system that uses the herbicide-resistant CP4-EPSPS gene as a selection marker was developed. Notably, all of the plants that were identified by PCR as transformants showed significant herbicide resistance. Additionally, this transformation protocol also highlighted: (i) the high yield of transgenic lines from calli (each gram of calli generated six transgenic lines); (ii) improved selection; and (iii) a higher transformation efficiency. This protocol provides a reliable tool for a routine procedure for the generation of resilient sugarcane plants.

Plant resistance against biotic stressors is significantly influenced by pathogenesis-related 1 (PR1) proteins. This study examines the systematic identification and characterization of PR1 family genes in sugarcane (Saccharum spontaneum Np-X) and the transcript expression of selected genes in two sugarcane cultivars (ROC22 and Zhongtang3) in response to Ustilago scitaminea pathogen infection. A total of 18 SsnpPR1 genes were identified at the whole-genome level and further categorized into four groups. Notably, tandem and segmental duplication occurrences were detected in one and five SsnpPR1 gene pairs, respectively. The SsnpPR1 genes exhibited diverse physio-chemical attributes and variations in introns/exons and conserved motifs. Notably, four SsnpPR1 (SsnpPR1.02/05/09/19) proteins displayed a strong protein–protein interaction network. The transcript expression of three SsnpPR1 (SsnpPR1.04/06/09) genes was upregulated by 1.2–2.6 folds in the resistant cultivar (Zhongtang3) but downregulated in the susceptible cultivar (ROC22) across different time points as compared to the control in response to pathogen infection. Additionally, SsnpPR1.11 was specifically upregulated by 1.2–3.5 folds at 24–72 h post inoculation (hpi) in ROC22, suggesting that this gene may play an important negative regulatory role in defense responses to pathogen infection. The genetic improvement of sugarcane can be facilitated by our results, which also establish the basis for additional functional characterization of SsnpPR1 genes in response to pathogenic stress.

Sugarcane mosaic virus (SCMV) (genus, Potyvirus; family, Potyviridae) is widespread, deleterious, and the most damaging pathogen of sugarcane (Saccharum officinarum L. and Saccharum spp.) that causes a substantial barrier to producing high sugarcane earnings. Sugarcane mosaic disease (SCMD) is caused by a single or compound infection of SCMV disseminated by several aphid vectors in a non-persistent manner. SCMV has flexuous filamentous particle of 700–750 nm long, which encapsidated in a positive-sense, single-stranded RNA molecule of 9575 nucleotides. RNA interference (RNAi)-mediated antiviral innate immunity is an evolutionarily conserved key biological process in eukaryotes and has evolved as an antiviral defense system to interfere with viral genomes for controlling infections in plants. The current study aims to analyze sugarcane (Saccharum officinarum L. and Saccharum spp.) locus-derived microRNAs (sof-miRNAs/ssp-miRNAs) with predicted potential for targeting the SCMV +ssRNA-encoded mRNAs, using a predictive approach that involves five algorithms. The ultimate goal of this research is to mobilize the in silico- predicted endogenous sof-miRNAs/ssp-miRNAs to experimentally trigger the catalytic RNAi pathway and generate sugarcane cultivars to evaluate the potential antiviral resistance surveillance ability and capacity for SCMV. Experimentally validated mature sugarcane (S. officinarum, 2n = 8X = 80) and (S. spp., 2n = 100–120) sof-miRNA/ssp-miRNA sequences (n = 28) were downloaded from the miRBase database and aligned with the SCMV genome (KY548506). Among the 28 targeted mature locus-derived sof-miRNAs/ssp-miRNAs evaluated, one sugarcane miRNA homolog, sof-miR159c, was identified to have a predicted miRNA binding site, at nucleotide position 3847 of the SCMV genome targeting CI ORF. To verify the accuracy of the target prediction accuracy and to determine whether the sugarcane sof-miRNA/ssp-miRNA could bind the predicted SCMV mRNA target(s), we constructed an integrated Circos plot. A genome-wide in silico-predicted miRNA-mediated target gene regulatory network was implicated to validate interactions necessary to warrant in vivo analysis. The current work provides valuable computational evidence for the generation of SCMV-resistant sugarcane cultivars.

Various RNA viral diseases on sugarcane result in yield loss and decreased sugar content. Breeding new varieties with virus resistance is the main goal of the sugarcane breeding program. Both single-stranded and double-stranded RNA viruses generated a double-stranded RNA replicative form (RF) during the replication cycle progress. While double-stranded RNA-specific ribonuclease (PAC1) encoded by the Pac1 gene (from Schizosaccharomyces pombe) can recognize and degrade double-stranded RNA specifically without any sequence, the expression of PAC1 in transgenic sugarcane may successfully develop virus-resistant sugarcane. In this research, we first expressed the PAC1 in prokaryotic cells. Then, double-stranded RNA RF of sugarcane's streak mosaic virus (SCSMV) was artificially synthesized. The degradation activity of the PAC1 was successfully tested by mixing the PAC1 protein and the double-stranded RNA RF. After that, the Pac1 gene was ligated to a plant expression vector and was then introduced into a virus-sensitive sugarcane cultivar by using Agrobacterium tumefaciens-mediated transformation method. Transgenic plants were challenged by inoculating with SCSMV. Results showed that although all the transgenic lines were infected by SCSMV, the mosaic symptoms that appeared on the leaves were significantly milder than that of the wild type. All transgenic shoots showed significantly lower viral loads and attained greater heights than wild-type shoots. This research provided a new pathway for breeding new varieties of sugarcane with virus resistance.

Sugarcane is a complex polyploid aneuploid cash crop, and transgenic varieties are important for molecular genetic and traditional breeding approaches. Herein, the sugarcane variety ROC22 served as the receptor, the Bar gene served as a screening marker, and positive and negative fragments of the ScD27.2 gene, upstream of strigolactones (SLs) biosynthesis genes driven by the 35S promoter, were introduced by Agrobacterium tumefaciens-mediated transformation. Regenerated plants were obtained by co-culture, screening culture, and differentiation induction, and 27 sense and antisense ScD27.2 transgenic seedlings were obtained by herbicide screening. PCR detection and 1% Basta (Glufosinate) application on leaves revealed Bar in all lines, with all testing positive for herbicide application and 23 containing the target gene (positive resistance screening rate = 87.5%). q-PCR and phenotypic analyses showed that ScD27.2 expression, plant height, tiller number, root length, stem diameter, and fresh weight were decreased in transgenic (ScD27.2R-9) compared with non-transgenic (NT and ScD27.2F-2) lines. ScD27.2 expression was downregulated, and growth potential was inhibited. Under 20% PEG treatment, malondialdehyde (MDA) content in ScD27.2R-9 was higher than in NT, while proline content was lower. Under drought stress, ScD27.2 expression, MDA levels, and proline content in ScD27.2F-2 and NT were higher than in non-treated controls, ScD27.2 expression increased with time, and MDA and proline levels also increased. ScD27.2 expression in ScD27.2R-9 decreased under 20% PEG treatment, MDA and proline increased (but not to NT levels), and growth was lower than NT. The 20% PEG treatment also increased the levels of (±)-2′-epi-5-deoxystrigol and (+)-abscisic acid in the rooting culture media of ScD27.2F-2, ScD27.2R-9, and NT lines, but the levels of (+)-abscisic acid content in ScD27.2R-9 was lower than in NT. Thus, interfering with ScD27.2 expression decreased resistance to 20% PEG treatment. ScD27.2 encodes a β-carotene isomerase involved in SLs biosynthesis that might function in sugarcane resistance to drought stress. It explains the role of SLs in sugarcane growth and development and responses to drought stress.

This study aimed to prepare the sugar industry for the possible introduction of genetically modified (GM) sugarcane and derived retail sugar products and to address several potential public concerns regarding the characteristics and safety of these products. GM sugarcane lines with integrated Cry1Ab and EPSPS foreign genes were used for GM sugar production. Traditional PCR, real-time fluorescent quantitative PCR (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed in analyzing leaves, stems, and other derived materials during sugar production, such as fibers, clarified juices, filter mud, syrups, molasses, and final GM sugar product. The toxicity of GM sugar was examined with a feeding bioassay using Helicoverpa armigera larvae. PCR and RT-qPCR results showed that the leaves, stems, fibers, juices, syrups, filter mud, molasses, and white granulated sugar from GM sugarcane can be distinguished from those derived from non-GM sugarcane. The RT-qPCR detection method using short amplified product primers was more accurate than the traditional PCR method. Molecular analysis results indicated that trace amounts of DNA residues remain in GM sugar, and thus it can be accurately characterized using molecular analysis methods. ELISA results showed that only the leaves, stems, fibers, and juices sampled from the GM sugarcane differed from those derived from the non-GM sugarcane, indicating that filter mud, syrup, molasses, and white sugar did not contain detectable Cry1Ab and EPSPS proteins. Toxicity analysis showed that the GM sugar was not toxic to the H. armigera larvae. The final results showed that the GM sugar had no active proteins despite containing trace amounts of DNA residues. This finding will help to pave the way for the commercialization of GM sugarcane and production of GM sugar.